IGEM:British Columbia/2009/Notebook/Biosensor Sensitivity/2009/07/29

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cPCR of Lock/Key/Control-lock into Ampicillin and Chloramphenicol construction plasmids (from July 28) to verify constructs

  • performed using standard UBC iGEM cPCR protocol (cultures were in triplicate)
  • note: this set of cPCR was done using colonies transformed with the ligation product that did not have heat-inactivated restriction endonucleases added to it
  • primers

Forward (FW): VF2 Reverse (RE): VR2

  • reagents for each reaction (μL)
  • 10x reaction buffer: 2.5
  • 10μM Forward primer: 1.25
  • 10μM Reverse primer: 1.25
  • 10mM dNTP: 0.5
  • Taq polymerase: 0.5
  • sdH20: 19.3
  • liquid culture: 0 (transferred using autoclaved wooden stick from plate)
  • PCR steps [temperature | time]
  • Initial denaturation: 94°C | 120s
  • Denaturation: 94°C | 30s
  • Annealing: 56°C | 30s
  • Extension: 72°C | 24s
  • Final extension: 72°C | 72 s
  • Step 2-4 repeated 24 cycles

Gel electrophoresis

  • performed using standard UBC iGEM protocols
  • at least band from each construct with the correct size; the corresponding cultures will be miniprepped tomorrow

plates from July 28

Plates with heat inactivation were sealed (parafilm) and stored at 4°C. Same with the transformants without heat inactivation of digest.

Making Minimal Media

  • The slow observance of RFP and GFP in the cells containing pBAD promoters may be from the cells preferentially chosing to take in glucose before arabinose.
  • To test this theory, M9 Minimal Media was made by following the protocol given on the M9 Minimaum Media page. Arabinose was used as the carbon source

NOTE: Two batches of M9 Salts were made. The first batch was made using anhydrous Na2HPO4. In order to keep the molarity of the final solution the same, 33g of anhydrous Na2HPO4 was used instead of 64g Na2HPO4.7H2O. The second batch of M9 salts were made with correctly hydrated salts.

NOTE: 3 batches of M9 Minimal Media was made. The first (M9 Minimal Media ‘A’), had non filter-sterilized arabinose added to it and was autoclaved at the end to sterilize it. After autoclaving, the arabinose caramilized and the solution turned a light brown. There was also some precipitation in the bottom of the flask. The second (M9 Minimal Media ‘B’) was made with the first batch of M9 salts containing anhydrous Na2HPO4. The third (M9 Minimal Media ‘C’) was made with the other batch of M9 Salts containing correctly hydrated salts.

Overnights with M9 Minimal Media

  • To test the ability of M9 Minimal Media to support growth
  • Overnights of J23100, pBAD(wt)+GFP and pBAD(weak)+GFP were started in 3 mL of each of the three batches (A, B and C) of M9 Minimal Media as well as LB-chlor+1% arabinose as a control

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